Neuronal ceroid lipofuscinoses (NCL) is a set of devastating inherited neurological diseases of infants, children and adults. There are at least 5 distinct human diseases. NCL also occurs in sheep and dogs. At this time little is known about the pathogenesis of NCL in any species. It is known that in all forms of NCL, lysosomes in many tissues store a hydrophobic proteolipid, subunit c of ATP synthase. Why this material is stored is not known. However, storage leads, by unknown mechanisms, to neuronal and usually to retinal degeneration. During the first 3 years of this grant, we have described the first mouse model of NCL, called mnd and subsequently discovered two other genetically distinct mouse models of NCL, ncl2 and ncl3. In addition we have also made the important discovery that a second proteolipid, vacuolar H+ATPase is also stored in lysosomes in dogs, sheep, all three mouse models, and perhaps in the juvenile form of NCL, Batten disease. In this application, we will utilize our mouse models to study the disease mechanisms of NCL. Specific Aim A tests the hypothesis that the storage of proteolipids is the direct cause of neuronal degeneration, and not simply a byproduct of abnormal cellular metabolism. We will test this by determining if the amount of proteolipid stored in each NCL mutant is directly correlated with the neuronal degeneration, and if the kind of degeneration is the same in each mutant. If the pathology is the same in all mutants, then that supports the hypothesis that storage of proteolipids leads directly to degeneration. In Specific Aim B we will genetically map ncl2 and ncl3 to within 2 centimorgans, and will test candidate genes for NCL that map to the same chromosomal location. This might lead to the identification of the first NCL gene in any species. In Specific Aim C we will use pulse chase biochemical techniques to determine if the accumulation of both proteolipids in cultured cells from ncl3/ncl3 mice is due to either overexpression or defective degradation of the proteolipid. This is the first step in elucidation of the mechanism of storage of proteolipids. We will also test the hypothesis that accumulation of vacuolar protein leads to accumulation of ATP synthase and to cellular degeneration. We will insert a tetracycline-controlled plasmid containing vacuolar protein into HeLa and PC12 cells. We will thereby be able to cause the cells to overexpress large amounts of vacuolar protein to study the biochemical responses, that may cause the cells to degenerate.